Hartmut Greiner

A pharmacological profile of the novel, peripherally-selective k-opioid receptor agonist, EMD 61753

1 The pharmacological properties of the novel diarylacetamide k‐opioid receptor agonist, EMD 61753, have been compared with those of ICI 197067 (a centrally‐acting k agonist) and ICI204448 (a peripherally‐selective k agonist). 2 EMD 61753 binds with high affinity (IC50 5.6 nm) and selectivity (k:μ:δ:σ binding ratio 1:536: 125: > 1,786) to k‐opioid receptors and is a full and potent (IC50 54.5 nm) agonist in an in vitro assay for k‐opioid receptors (rabbit vas deferens preparation). 3 Systemically‐applied [14C]‐EMD 61753 is found in high concentrations in the lungs, liver, adrenal glands and kidneys. Considerably less radioactivity is detected in the whole brain, and this radioactivity is concentrated in the region of the cerebral ventricles in the choroid plexuses. EMD 61753 penetrates only poorly into the CNS. 4 EMD 61753 was weakly effective in pharmacological tests of central activity. This compound reversed haloperidolol‐induced DOPA accumulation in the nucleus accumbens of the rat only at a dose of 30mg kg−1, s.c., (doses of 0.1, 1.0 and 10mg kg−1, s.c., and 1.0, 10 and 100 mg kg−1, p.o., were inactive). Hexobarbitone‐induced sleeping in mice was prolonged by EMD 61753 at threshold doses of 10 mg kg−1, s.c., and 100 mg kg−1, p.o., whereas the motor performance of rats in the rotarod test was impaired by EMD 61753 with an ID50 value of 453 mg kg−1, s.c. 5 EMD 61753 produced dose‐dependent, naloxone‐reversible antinociception in the mouse formalin test (1st phase ID50 1.9 mg kg−1, s.c., and 10.4 mg kg−1, p.o.; 2nd phase ID50 0.26 mg kg−1, s.c., and 3.5 mg kg−1 p.o.) and rodent abdominal constriction test (ID50 mouse 1.75 mg kg−1, s.c., and 8.4 mg kg−1, p.o.; ID50 rat 3.2 mg kg−1, s.c., and 250 mg kg−1, p.o.). EMD 61753 was inactive, or only weakly effective, in the rat pressure test under normalgesic conditions. After the induction of hyperalgesia with carrageenin, however, this compound elicited potent, dose‐dependent (ID50 0.08 mg kg−1, s.c., and 6.9 mg kg−1, p.o., after remedial application, and 0.2 mg kg−1, s.c., and 3.1 mg kg−1, p.o., after prophylactic application) and naloxone‐reversible antinociception. The antinociceptive action of systemically‐applied (50 mg kg−1, p.o.) EMD 61753 in the hyperalgesic pressure test was completely inhibited by injection of the k‐opioid antagonist norbinaltorphimine (100 μg) into the inflamed tissue, a result which indicates that this opioid effect is mediated peripherally. 6 Cutaneous plasma protein extravasation produced by antidromic electrical stimulation of the rat saphenous nerve was dose‐dependently inhibited by systemically‐applied EMD 61753 (ID50 values 3.7 mg kg−1, s.c., and 35.8 mg kg−1, p.o.), and this effect was completely antagonized by intraplantar application of norbinaltorphimine (50 μg). Extravasation elicited by the intraplantar application of substance P (10 μg) was not influenced by the administration of EMD 61753. 7 EMD 61753 produced dose‐dependent diuresis in non‐hydrated rats at doses of and above 10 mg kg−1, s.c., and 10 mg kg−1, p.o., and in saline‐loaded rats at doses of and above 10 mg kg−1, s.c., and 30 mg kg−1, p.o. 8 The prostaglandin‐mediated fall in mean arterial blood pressure elicited in anaesthetized rats by i.v. application of arachidonic acid was not inhibited by prior treatment with EMD 61753 (lO mg kg−1, p.o.). Thus, a blockade of prostaglandin synthesis via inhibition of cyclo‐oxygenase activity does not contribute to the in vivo effects of EMD 61753 and its metabolites. 9 The present experiments therefore indicate that EMD 61753 is a potent, selective and orally‐effective full K‐opioid receptor agonist which has a limited ability to penetrate the blood‐brain barrier and elicit centrally‐mediated sedation, putative aversion, diuresis, and antinociception. The inhibitory actions of systemically‐applied EMD 61753 against hyperalgesic pressure nociception and neurogenic inflammation are mediated peripherally, probably by opioid receptors on the endings of sensory nerve fibres.

Biochemical and functional studies on EMD 49,980: a potent, selectively presynaptic D-2 dopamine agonist with actions on serotonin systems.

EMD 49,980 proved to be a potent and selectively presynaptic D-2 dopamine receptor agonist in biochemical studies with rats. Thus, the gamma-butyrolactone-induced accumulation of dihydroxyphenylalanine, used as a presynaptic model, was antagonized with ED50 values of 0.29 and 0.09 mumol/kg in striatum and t. olfactorium, respectively, with high maximal effects. In contrast, striatal acetylcholine concentrations, reflecting actions at normosensitive postsynaptic D-2 receptors, were only moderately increased by about 30% over a dose range of 2.3-68 mumol/kg. In rats with unilateral nigrostriatal lesions, EMD 49,980 induced long-lasting contralateral turning, indicative of agonistic actions at denervated postsynaptic D-2 receptors. In addition, EMD 49,980 potently inhibited serotonin (5-HT) uptake in vitro and in vivo. Binding studies confirmed D-2 activity in the nM range but, similarly potent effects were observed at 5-HT1A binding sites. Measurement of 5-hydroxytryptophan (5-HTP) accumulation in the n. raphe revealed that, in vivo, the net effect of EMD 49,980 on 5-HT systems is an agonistic one. Control experiments indicate that inhibition of 5-HTP accumulation by EMD 49,980 is induced mainly via direct activation of 5-HT1A receptors, although some contribution due to 5-HT uptake inhibition is likely. Furthermore, results with various reference compounds make it unlikely that there are indirect effects, also via alpha 2-receptors in the models used and support the view that D-2 agonistic, 5-HT uptake inhibiting and 5-HT1A agonistic actions are independent properties of EMD 49,980.

Fragment-based discovery of new highly substituted 1H-pyrrolo[2,3-b]- and 3H-imidazolo[4,5-b]-pyridines as focal adhesion kinase inhibitors.

Focal adhesion kinase (FAK) is considered as an attractive target for oncology, and small-molecule inhibitors are reported to be in clinical testing. In a surface plasmon resonance (SPR)-mediated fragment screening campaign, we discovered bicyclic scaffolds like 1H-pyrazolo[3,4-d]pyrimidines binding to the hinge region of FAK. By an accelerated knowledge-based fragment growing approach, essential pharmacophores were added. The establishment of highly substituted unprecedented 1H-pyrrolo[2,3-b]pyridine derivatizations provided compounds with submicromolar cellular FAK inhibition potential. The combination of substituents on the bicyclic templates and the nature of the core structure itself have a significant impact on the compounds FAK selectivity. Structural analysis revealed that the appropriately substituted pyrrolo[2,3-b]pyridine induced a rare helical DFG-loop conformation. The discovered synthetic route to introduce three different substituents independently paves the way for versatile applications of the 7-azaindole core.

Central and peripheral actions of the novel κ-opioid receptor agonist, EMD 60400

1 The pharmacological characteristics of the κ‐opioid receptor agonist, EMD 60400, have been investigated, with particular reference to its central and peripheral sites of action and its ability to influence nociception. The κ agonists ICI 197067 and ICI 204448 were tested for purposes of comparison. 2 EMD 60400 and ICI 197067 bind with high affinity (IC50 values of 2.8 and 1.5 nm, respectively) and high selectivity to κ‐opioid receptors. ICI 204448 has a lower binding affinity (IC50 13.0 nm) and selectivity for κ‐opioid receptors. 3 EMD 60400, ICI 197067, and ICI 204448 are full and potent agonists in the rabbit vas deferens in vitro assay for κ‐opioid receptors (IC50 values of 41.8, 15.7 and 15 nm, respectively). 4 Ex vivo binding experiments in mice revealed that EMD 60400 and ICI 197067 were well taken up after s.c. administration. Brain levels of EMD 60400 were lower than those of ICI 197067 at comparable doses, indicating that EMD 60400 does not penetrate into the CNS as well as ICI 197067. 5 Haloperidol‐induced DOPA accumulation in the nucleus accumbens of the rat was dose‐dependently reversed by s.c. application of EMD 60400 and ICI 197067 at doses of and above 3 and 0.3 mg kg−1, respectively. ICI 204448 had no effect on DOPA accumulation at 30 mg kg−1, s.c. 6 Prolongation of hexobarbitone‐induced sleeping time in mice and motor impairment in the rat rotarod test were observed for EMD 60400 at doses above 3 and 2.5 mg kg−1, s.c., respectively, and for ICI 197067 at doses above 0.3 and 0.25 mg kg−1, s.c., respectively. ICI 204448 was inactive in these tests at doses of 30 and 100 mg kg−1, s.c., respectively. 7 EMD 60400 applied s.c. produced dose‐dependent naloxone‐reversible antinociception in the mouse formalin test (1st and 2nd phase ID50 0.44 and 0.47 mg kg−1, respectively) and rodent writhing test (ID50 mouse 0.55 mg kg−1 and rat 0.3 mg kg−1). Furthermore, EMD 60400 was considerably more potent in the rat pressure pain test after the induction of inflammation with carrageenin than under normalgesic conditions (ID50 values 0.1 μg kg−1 and 4.0 mg kg−1, s.c., respectively). The action of EMD 60400 (50 μg kg−1, s.c.) in the hyperalgesic pressure pain test was completely antagonized by injection of the κ‐opioid antagonist, norbinaltorphimine (100 μg) into the inflamed tissue, thus demonstrating the peripheral opioid nature of this effect. 8 EMD 60400 produced dose‐dependent inhibition of neurogenic plasma extravasation elicited by antidromic electrical stimulation of the rat saphenous nerve (ID50 value 0.3 mg kg−1, i.v.). This inhibition was completely antagonized by intraplantar injection of norbinaltorphimine (50 μg). 9 EMD 60400, ICI 197067, and ICI 204448 have diuretic effects in rats at doses of and above 0.1, 0.01, and 0.3 mg kg−1, s.c., respectively. An antidiuretic action was also observed with ICI 197067 at very low doses (3 and 6 μg kg−1, s.c.). 10 Pharmacological and biochemical data therefore indicate that the three κ‐opioid receptor agonists tested here have different tendencies to elicit centrally‐mediated sedation and putative aversion (ICI 197067 > EMD 60400 > ICI 204448) which correspond to their ability to cross the blood‐brain barrier. EMD 60400 combines high affinity and selectivity for the κ receptor with a degree of peripheral selectivity. The peripheral actions of systemically‐applied EMD 60400 against hyperalgesic pressure pain and neurogenic inflammation are very probably mediated by opioid receptors on the endings of sensory nerve fibres.

EMD 61 753 as a favourable representative of structurally novel arylacetamido-type K opiate receptor agonists

Abstract K opiate agonists like (−)-U 50488H, (−)-PD 117302, etc. contain an acetamido group which is mono substituted in the α-position by an aromatic moiety. In contrast, EMD 61 753 is di substituted in this position by two phenyl rings and is thus the first representative of the new class of diarylacetamide-type κ opiates. Derivatives of EMD 61 753 are described and structure-activity relationships are discussed. In the formalin test in mice EMD 61 753 shows a profile similar to that of the antiinflammatory drugs rather than that of the centrally acting opiates.

Selection of fragments for kinase inhibitor design: decoration is key.

In fragment-based screening, the choice of the best suited fragment hit among the detected hits is crucial for success. In our study, a kinase lead compound was fragmented, the hinge-binding motif extracted as a core fragment, and a minilibrary of five similar compounds with fragment-like properties was selected from our proprietary compound database. The structures of five fragments in complex with transforming growth factor β receptor type 1 kinase domain were determined by X-ray crystallography. Three different binding modes of the fragments are observed that depend on the position and the type of the substitution at the core fragment. The influence of different substituents on the preferred fragment pose was analyzed by various computational approaches. We postulate that the replacement of water molecules leads to the different binding modes.

Bioisosterism of fluorine and cyano as indole substituents. Theoretical, in vitro and in vivo examination

Fluorine substitution modifies structural attributes and often induces unexpected effects. When substituting fluorine with cyano in position 5 of the indole in indole-butyl-amines, the properties of the compounds proved to be very comparable. In vitro target-profile, metabolism, and in vivo activity in the ultrasonic vocalisation test indicate bioisosterism between the two substituents.